Remifentanil was obtained from GlaxoSmithKline Pharmaceuticals (Rockville, MD, USA) and dissolved in research-grade deionized distilled water (DNAse- and RNAse-free). M-CSF and recombinant RANKL were purchased from PeproTech (Rocky Hill, NJ, USA). Antibodies against c-fos and nuclear factor of activated T cell cytoplasmic 1 (NFATc1) were obtained from SantaCruz Biotechnology (Santa Cruz, CA, USA). The Leukocyte Acid Phosphatase (TRAP) kit and anti-β-actin antibody were obtained from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals and reagents were purchased from Sigma-Aldrich.

BMMs were isolated from mice bone marrow and used as osteoclast precursor cells for in vitro osteoclast differentiation, as previously described [16]. Mouse whole bone marrow cells were collected from the femurs and tibiae of 5-week-old ICR mice, placed in 100 mm dishes, and cultured overnight in α-modified Eagle Medium (α-MEM; WelGENE Inc., Daegu, Korea) supplemented with 10% fetal bovine serum (FBS). Adherent stromal cells were discarded and the floating cells were cultured in the presence of M-CSF (30 ng/ml) in Petri dishes. The cells became adherent after 3–4 days of culture and were used as BMMs, the osteoclast precursor cells. Animal experiments were approved by the Committees on the Care and Use of Animals in Research at Pusan National University. To induce osteoclast differentiation, BMMs (4 × 10⁴ cells/48-well plates) were cultured with osteoclastogenic medium (OM: 30 ng/ml M-CSF + 100 ng/ml RANKL) for 4 days. After culture, the cells were stained for TRAP activity and the TRAP-positive multinucleated cells with ≥ 3 nuclei were considered to be osteoclasts.

The differentiation of pre-OCs, mononuclear TRAP-positive cells, was induced through the culture of BMMs with OM for 2 days. The pre-OCs were treated with fresh α-MEM (with M-CSF and RANKL) in the presence or absence of various concentrations of remifentanil and cultured for up to 3 days (5 days in total). All experiments were conducted on pre-OCs.

Cytotoxicity measurement and cell proliferation assay The effects of remifentanil on both cell viability and proliferation were examined by the well-established colorimetric 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich) assay. In brief, pre-OCs were seeded in 96-well plates with the indicated concentrations of remifentanil (0.1, 1, 10, and 100 ng/ml) for up to 3 days. At the end of the culture period, the cells were incubated with MTT solution (medium containing 0.5 mg/ml MTT) for a further 4 h. After culture, the formation of the blue formazan product was measured at 570 nm by using a microplate reader.

The cells were fixed in 3.7% formalin for 10 min, washed twice with phosphate buffered saline (PBS), and permeabilized by the addition of 0.3 mg/ml Fast Red Violet LB in 0.05 M sodium acetate, 0.05 M acetic acid, 0.03 M sodium tartrate, 0.1 mg/ml naphthol AS-MX phosphate disodium salt, and 0.1% Triton X-100 for 1 min. The cells were the washed with PBS and stained for TRAP activity by using a TRAP kit (Sigma) in accordance with the manufacturer's instructions. After incubation at 37℃ for 1 h, the cells were washed three times with distilled water. TRAP-positive cells appeared red or purple.

Total RNA was isolated by using TRIzol reagent (Invitrogen, CA, USA) and 1.5 µg was reverse-transcribed by using Superscript II (Invitrogen) in accordance with the manufacturer's protocol. For real-time PCR analysis, 2 µg of cDNA was amplified with SYBR green PCR master mix (Applied Biosystems) for 40 cycles of 15 s at 95℃ for denaturation and 1 min at 60℃ for amplification by using an AB7500 (Applied Biosystems). Relative mRNA expression levels were obtained after normalizing against actin mRNA levels. The primer sets used in real-time PCR were as follows: NFATc1, 5′-CCA GTA TAC CAG CTC TGC CA-3′ (forward) and 5′-GTG GGA AGT CAG AAG TGG GT-3′ (reverse); c-fos, 5′-ACT TCT TGT TTC CGG C-3′ (forward) and 5′-AGC TTC AGG GTA GGT G-3′ (reverse); DC-STAMP, 5′-GGG TGC TGT TTG CCG CTG-3′ (forward) and 5′-CGA CTC CTT GGG TTC CTT GCT-3′ (reverse); CTK, 5′-ATA TGT GGG CCA CCA TGA AAG TT-3′ (forward) and 5′-TCG TTC CCC ACA GGA ATC TCT-3′ (reverse); CTR, 5′-GCA ACC GAA CCT GGT CCA ACT AT-3′ (forward) and 5′-AAG CAG CAA TCG ACA AGG AGT GA-3′ (reverse); Actin, 5′-TCT GGC ACC ACA CCT TCT AC-3′ (forward) and 5′-TAC GAC CAG AGG CAT ACA GG-3′ (reverse).

Transwells with 8 µm porosity polycarbonate membrane inserts were used. Pre-OCs were suspended in α-MEM media at 1×10⁵ cells/100 µl and were added to the upper chamber. Remifentanil (10 and 50 ng/ml) in α-MEM media was added into the lower chamber. Pre-OCs that migrated through the filter and appeared on the lower side were fixed by careful immersion of the filter into methanol for 1 min, stained with hematoxylin and eosin (H&E) solution, and counted in three random fields per well. Each experiment was performed in duplicate and three separate experiments were performed for each treatment group.

Data were obtained from at least three independent experiments. Student's t-test was used to determine the significance of differences between two groups. Differences with P < 0.01 were regarded as statistically significant and denoted with an asterisk (^**P < 0.01; ^***P < 0.001).

BMMs were cultured with OM for 2 days to induce differentiation to pre-OCs (Fig. 1A). The effect of remifentanil on the cell viability and proliferation of pre-OCs was verified by using an MTT assay. The MTT assay indicated that the cell viability of pre-OCs was not affected by treatment with various concentrations of remifentanil (0.1, 1, 10, and 100 ng/ml) (Fig. 1B). The proliferation of pre-OCs cultured in OM for 3 days with remifentanil was maintained and no significant difference in cell proliferation was identified at different remifentanil concentrations (Fig. 1B).

Pre-OCs were cultured with M-CSF alone or OM in the presence of remifentanil for 2–3 days to evaluate the formation of mature osteoclasts. TRAP staining was used for this analysis; TRAP-positive multinucleated cells with ≥ 3 nuclei were considered to be mature osteoclasts. Remifentanil increased the formation of TRAP-positive multinucleated osteoclasts, especially after 3 days (Fig. 2A). The size of osteoclasts was significantly increased by the presence of remifentanil (50 ng/ml) compared with the absence of remifentanil (Fig. 2B). These results confirmed that the administration of remifentanil to pre-OCs enhanced osteoclast differentiation and maturation.

NFATc1, a member of the NFAT family of transcription factor genes [17], is known as key transcription factor that induces a number of genes involved in osteoclast differentiation. It is strongly induced following RANKL stimulation. The induction of NFATc1 following RANKL is regulated by intracellular calcium oscillation and another transcription factor, c-fos [1718]. To investigate the effect of remifentanil on the expression of transcription factors such as NFATc1 and c-fos, the relative mRNA expression of NFATc1 and c-fos genes from pre-OCs treated with M-CSF alone or M-CSF and RANKL for 2 and 4 days was measured by using quantitative real-time PCR. The relative mRNA expression of NFATc1 and c-fos in pre-OCs was not altered by an increase in the dose of remifentanil (0, 10, and 50 ng/ml) during osteoclastogenesis (Fig. 3A, B). These results confirmed that the enhancement of pre-OC differentiation by remifentanil was not mediated by the activation of NFATc1 and c-fos.

TRAP, CTK, and CTR are osteoclast differentiation marker genes that are induced by NFATc1 activation at the final stage of differentiation [19]. As previous results showed that remifentanil upregulated pre-OC differentiation (Fig. 2A, B), we hypothesized that remifentanil may increase the expression of osteoclast-specific genes. The mRNA expression of osteoclastogenic-specific genes was analyzed in pre-OCs. As shown in Fig. 4A, B, and C, the relative mRNA expression of TRAP, CTK, and CTR was not significantly increased for remifentanil treatment (10 and 50 ng/ml) compared with the presence of only M-CSF and RANKL on days 2 and 4.

DC-STAMP is a key regulator in osteoclast cell-cell fusion, which is a critical process for osteoclast maturation [20]. To investigate the relationship of cell-cell fusion with the upregulation of pre-OC differentiation induced by remifentanil, the mRNA expression of DC-STAMP was evaluated. Treatment with remifentanil increased the relative mRNA expression of DC-STAMP in a dose-dependent manner after 4 days, but the difference was not significant (Fig. 4D).

The migration of pre-OCs was evaluated by using the Boyden chamber assay. Pre-OCs in medium were seeded in the upper part of a Transwell plate and allowed to migrate vertically through the pores of the membrane into the lower chamber, which held the control or remifentanil-containing medium. The number of migrated cells was increased after remifentanil treatment (10 and 50 ng/ml) in the lower chamber compared with the control (Fig. 5A). In addition, the number of migrated cells was significantly increased by 50 ng/ml remifentanil compared with the untreated control (Fig. 5B).